A study of differential circRNA and lncRNA expressions in COVID-19-infected peripheral blood.

To conquer the worldwide outbreak of COVID-19 virus, a large number of studies have been carried out on COVID-19 infection, transmission and treatment. However, few studies have been conducted from the perspectives of circRNA and lncRNA, which are known to be involved in regulating many life activities, such as immune tolerance and immune escapes, and hence may provide invaluable information in the emerging COVID-19 infection and recurrence. Moreover, exosomes has been reported to play an important role in COVID-19 recurrence, and thus may interact with the expression of circRNA and lncRNA. In this work, we sequenced circRNA, lncRNA and mRNA from recurrent COVID-19 patients and healthy people, and compared the differences. GO and KEGG enrichment analysis show that differentially expressed circRNA and lncRNA are mainly involved in the regulation of host cell cycle, apoptosis, immune inflammation, signaling pathway and other processes. The comparison to exosomes related databases shows that there are 114 differentially expressed circRNA, and 10 differentially expressed lncRNA related to exosomes. These studies provide reference for exploring circRNA and lncRNA to study the infection mechanism of COVID-19, their diagnostic and therapeutic values, as well as the possibility to employ them as biomarkers.

AcMNPV-miR-3 is a miRNA encoded by Autographa californica nucleopolyhedrovirus and regulates the viral infection by targeting ac101.

MicroRNAs (miRNAs), which are small noncoding RNAs found in plants, animals, and many viruses, regulate various biological processes. Our group has previously reported the first miRNA encoded by Autographa californica multiple Nucleopolyhedrovirus (AcMNPV), AcMNPV-miR-1, which regulates the expression of three viral genes. This study characterizes another miRNA encoded by AcMNPV, AcMNPV-miR-3. This miRNA is located on the opposite strand of the viral gene ac101 coding sequence in the AcMNPV genome, and it can be detected at 6 h post-infection and accumulated to a peak around 12 h post-infection in AcMNPV infected Sf9 cells. Five viral genes (ac101, ac23, ac25, ac86, and ac98) were verified to be regulated by AcMNPV-miR-3. Ac101 was markedly down-regulated by AcMNPV-miR-3 that may be via a siRNA-like cleavage mode. Administrating excessive AcMNPV-miR-3 resulted in decreased production of infectious budded virions (BV) and accelerated the formation of occlusion-derived virions (ODV). These results suggest that AcMNPV-miR-3 may play a regulatory role in BV and ODV production.

Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication.

UNLABELLED: An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae. IMPORTANCE: Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a better general understanding of virus-encoded miRNAs. In brief, our study implied that AcMNPV-miR-1 constrains viral replication and cellular infection but enhances larval infection.

Autographa californica multiple nucleopolyhedrovirus gene ac81 is required for nucleocapsid envelopment.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly pathogenic Baculoviridae that targets insects, whose core gene, ac81, has an unknown function. To determine the role of ac81 in the life cycle of AcMNPV, an ac81-knockout (Ac-81KO-GP) was constructed through homologous recombination in Escherichia coli. We determined that no budded virions were produced in Ac-81KO-GP-transfected Sf9 cells, while there was no effect on viral DNA replication. Electron microscopy (EM) analysis revealed that occlusion-derived virions (ODVs) envelopment and the subsequent embedding of virions into occlusion bodies (OBs) were aborted due to ac81 deletion. Interestingly, confocal microscopy and immunofluorescence analysis revealed that Ac81 was predominantly localized to the ring zone of nuclei during the late phase of infection. In addition, Ac81 was localized to the mature and premature ODVs in virus-infected cells within the ring zone as revealed by immuno-electron microscopy (IEM) analysis. Furthermore, we determined that Ac81 contained a functional hydrophobic transmembrane (TM) domain, whose deletion resulted in a phenotype similar to that of Ac-81KO-GP. These results suggest that Ac81 might be a TM protein that played an important role in nucleocapsid envelopment.

COStar: a D-star Lite-based dynamic search algorithm for codon optimization.

Codon optimized genes have two major advantages: they simplify de novo gene synthesis and increase the expression level in target hosts. Often they achieve this by altering codon usage in a given gene. Codon optimization is complex because it usually needs to achieve multiple opposing goals. In practice, finding an optimal sequence from the massive number of possible combinations of synonymous codons that can code for the same amino acid sequence is a challenging task. In this article, we introduce COStar, a D-star Lite-based dynamic search algorithm for codon optimization. The algorithm first maps the codon optimization problem into a weighted directed acyclic graph using a sliding window approach. Then, the D-star Lite algorithm is used to compute the shortest path from the start site to the target site in the resulting graph. Optimizing a gene is thus converted to a search in real-time for a shortest path in a generated graph. Using in silico experiments, the performance of the algorithm was shown by optimizing the different genes including the human genome. The results suggest that COStar is a promising codon optimization tool for de novo gene synthesis and heterologous gene expression.

A microRNA encoded by Autographa californica nucleopolyhedrovirus regulates expression of viral gene ODV-E25.

Baculovirus-encoded microRNAs (miRNAs) have been described in Bombyx mori nucleopolyhedrovirus; however, most of their functions remain unclear. Here we report the identification and characterization of an miRNA encoded by Autographa californica nucleopolyhedrovirus. The identified miRNA, AcMNPV-miR-1, perfectly matched a segment in the coding sequence of the viral gene ODV-E25 and downregulated ODV-E25 mRNA expression, which likely resulted in a reduction of infectious budded virions and accelerated the formation of occlusion-derived virions.

metaPIS: a sequence-based meta-server for protein interaction site prediction.

The identification of interfaces in protein complexes is effective for the elucidation of protein function and helps us to understand their roles in biological processes. With the exponentially growing amount of protein sequence data, an exploration of new methods that predict protein interaction sites based solely on sequence information is becoming increasingly urgent. Because a combination of different methods could produce better results than a single method, interaction site prediction can be improved through the utilization of different methods. This paper describes a new method that predicts interaction sites based on protein sequences by integrating five different algorithms employing meta-method, Majority Vote and SVMhmm Regression techniques. The 'metaPIS' web-server was implemented for meta-prediction. An evaluation of the meta-methods using independent datasets revealed that Majority Vote achieved the highest average Matthews correlation coefficient (0.181) among all the methods assessed. SVMhmm Regression achieved a lower score but provided a more stable result. The metaPIS server allows experimental biologists to speculate regarding protein function by identifying potential interaction sites based on protein sequence. As a web server, metaPIS is freely accessible to the public at http://202.116.74.5:84/metapis.

Study on CCR5 analogs and affinity peptides.

The G protein-coupled receptor of human chemokine receptor 5 (CCR5) is a key target in the human immunodeficiency virus (HIV) infection process due to its major involvement in binding to the HIV type 1 (HIV-1) envelope glycoprotein gp120 and facilitating virus entry into the cells. The identification of naturally occurring CCR5 mutations (especially CCR5 delta-32) has allowed us to address the CCR5 molecule as a promising target to prevent or resist HIV infection in vivo. To obtain high-affinity peptides that can be used to block CCR5, CCR5 analogs with high conformational similarity are required. In this study, two recombinant proteins named CCR5 N-Linker-E2 and CCR5 mN-E1-E2 containing the fragments of the CCR5 N-terminal, the first extracellular loop or the second extracellular loop are cloned from a full-length human CCR5 cDNA. The recombinant human CCR5 analogs with self-cleavage activity of the intein Mxe or Ssp in the vector pTwinI were then produced with a high-yield expression and purification system in Escherichia coli. Experiments of extracellular epitope-activity identification (such as immunoprecipitation and indirective/competitive enzyme-linked immunosorbent assay) confirmed the close similarity between the epitope activity of the CCR5 analogs and that of the natural CCR5, suggesting the applicability of the recombinant CCR5 analogs as antagonists of the chemokine ligands. Subsequent screening of high-affinity peptides from the phage random-peptides library acquired nine polypeptides, which could be used as CCR5 peptide antagonists. The CCR5 analogs and affinity peptides elucidated in this paper provide us with a basis for further study of the mechanism of inhibition of HIV-1 infection.

Benefits of random-priming: exhaustive survey of a cDNA library from lung tissue of a SARS patient.

The severe acute respiratory syndrome (SARS) leads to severe injury in the lungs with multiple factors, though the pathogenesis is still largely unclear. This paper describes the particular analyses of the transcriptome of human lung tissue that was infected by SARS-associated coronavirus (SARS-CoV). Random primers were used to produce ESTs from total RNA samples of the lung tissue. The result showed a high diversity of the transcripts, covering much of the human genome, including loci which do not contain protein coding sequences. 10,801 ESTs were generated and assembled into 267 contigs plus 7,659 singletons. Sequences matching to SARS-CoV RNAs and other pneumonia-related microbes were found. The transcripts were well classified by functional annotation. Among the 7,872 assembled sequences that were identified as from human genome, 578 non-coding genes were revealed by BLAST search. The transcripts were mapped to the human genome with the restriction of identity=100%, which found a candidate pool of 448 novel transcriptional loci where EST transcriptional signal was never found before. Among these, 13 loci were never reported to be transcriptional by other detection methods such as gene chips, tiling arrays, and paired-end ditags (PETs). The result showed that random-priming cDNA library is valid for the investigation of transcript diversity in the virus-infected tissue. The EST data could be a useful supplemental source for SARS pathology researches.

Discrimination of thermostable and thermophilic lipases using support vector machines.

Discriminating thermophilic lipases from their similar thermostable counterparts is a challenging task and it would help to design stable proteins. In this study, the distributions of N (N=2, 3) neighboring amino acids and the non-adjacent di-residue coupling patterns in the sequences of 65 thermostable and 77 thermophilic lipases had been systematically analyzed. It was found that the hydrophobic residues Leu, Pro, Met, Phe, Trp, as well as the polar residue Tyr had higher occurrence in thermophilic lipases than thermostable ones. The occurrence frequencies of KC EE KE RE, VE, YI, EK, VK, EV, YV, EY, KY, VY and YY in thermophilic proteins were significantly higher, while the occurrence frequencies of QC, QH, QN, HQ, MQ, NQ, QQ, TQ, QS and QT were significantly lower. CXP or CPX showed significantly positive to lipase thermostability, while XXQ or QXX showed significantly negative to lipase thermostability. Non-adjacent di-residue coupling patterns of PR14, RY32, YR47, LE53, LE64, PP64, RP70 and PP101 were significantly different in thermophilic lipases and their thermostable counterparts. The composition of dipeptide, tripeptide and non-adjacent di-residue patterns contained more information than amino acid composition. A statistical method based on support vector machines (SVMs) was developed for discriminating thermophilic and thermostable lipases. The accuracy of this method for the training dataset was 97.17?. Furthermore, the highest accuracy of the method for testing datasets was 98.41?. The influence of some specific patterns on lipase thermostability was also discussed.

Effect of ac68 knockout and lef3 leading sequence disruption on viral propagation.

Orf68 (ac68) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is identified to be an early gene, but its transcription start site remains unknown. The coding sequence of ac68 overlaps 280-bp leading sequence and 159-bp coding sequence of lef3 (ac67). In this study, the transcription start site of ac68 was determined by 5' RACE analysis to be 18 nucleotides upstream from the start codon. In order to investigate the effect of ac68 deletion on virus propagation, we generated a bacmid with an ac68 knockout by deleting 360-bp inside the ac68 gene, which also deleted 220-bp leading sequence of lef3. Production of infectious budded virus and formation of nucleocapsids and occlusion bodies exhibited wild-type patterns of virus propagation in Sf-9 cells infected with the mutant bacmid. The result demonstrated that ac68 was not an essential gene for viral propagation which was confirmed by further deletion of ac68, and disruption of the lef3 leading sequence did not affect viral propagation. Ac68 was the second auxiliary gene discovered besides Ac133 (alk-exo) among the 30 core genes of AcMNPV.

ac109 is required for the nucleocapsid assembly of Autographa californica multiple nucleopolyhedrovirus.

ORF109 (Ac109) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in all sequenced baculovirus genomes, but its function is not known. This paper describes generation of an ac109 knockout virus (Ac-ac109-KO-GP) and analyses of the influence of ac109 deletion on the virus replication in Sf-9 cells so as to investigate the role of ac109 in the viral life cycle. Results revealed that budded virus (BV) yields and occlusion body synthesis were completely blocked in cells infected with the mutant virus. Electron microscopy demonstrated that ac109 deletion blocked nucleocapsid formation, though infection was initiated and electron-dense bodies associated with the virogenic stroma appeared. The mutant phenotype was rescued by an ac109 rescue virus. On the other hand, real-time PCR analysis indicated that ac109 is not required for viral DNA replication. Thus, these results suggested that ac109 plays an important role in AcMNPV nucleocapsid formation.

Characterization of AcMNPV with a deletion of ac68 gene.

Orf68 (ac68) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene that codes a predicted 192-amino acid protein, but its function remains unknown. Results of the current study showed that ac68 was transcribed from 3 to 96 h and the protein was detected from 36 to 96 h post infection. An ac68 knockout bacmid was generated to investigate the role of the gene in baculovirus life cycle. Analyses of the production of infectious budded virus, occlusion bodies, and the formation of nucleocapsids revealed that there was no difference between the infection patterns of the mutant and its parent virus, or the ac68 repair virus. Bioassay with Trichoplusia ni demonstrated that deletion of ac68 did not affect AcMNPV infectivity, but extended LT(50) to a longer value. Taken together, our results indicated that the deletion did not affect viral propagation both in vitro and in vivo, but deletion of the gene may affect the virulence in T. ni larvae.

Serologic and genetic characterization analyses of a highly pathogenic influenza virus (H5N1) isolated from an infected man in Shenzhen.

Highly pathogenic avian influenza (HPAI) H5N1 virus caused a wave of outbreaks in China during 2005--2006, resulting in a total of 20 cases of human infection in 14 provinces of China. On June16, 2006, a case of H5N1 human infection was confirmed in Shenzhen. The virus isolated from the patient, A/Guangdong/2/06, was characterized genetically and the relationship between the tracheal virus load and the antibody titer of the infected man was analyzed. Serological analysis confirmed that the patient's neutralizating antibodies had been generated 2 weeks after the onset of symptoms. The patient's serum antibodies could efficiently neutralize A/Guandong/2/06 infectivity in vitro. Phylogenetic analysis showed that the H5N1 virus of Shenzhen belonged to subclade 2.3.4, which contained viruses that were mainly responsible for the outbreaks in domestic poultry and in the cases of human infection in southern China. Homology and molecular characterization analysis revealed that all the segments of Shenzhen H5N1 virus still belonged to avian segments. Several specific amino acid residue mutations were detected.

Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation.

Although orf66 (ac66) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in all sequenced lepidopteran baculovirus genomes, its function is not known. This paper describes generation of an ac66 knockout AcMNPV bacmid mutant and analyses of the influence of ac66 deletion on the virus replication in Sf-9 cells so as to determine the role of ac66 in the viral life cycle. Results indicated that budded virus (BV) yields were reduced over 99% in ac66-null mutant infected cells in comparison to that in wild-type virus infected cells. Optical microscopy revealed that occlusion body synthesis was significantly reduced in the ac66 knockout bacmid-transfected cells. In addition, ac66 deletion interrupted preoccluded virion synthesis. The mutant phenotype was rescued by an ac66 repair bacmid. On the other hand, real-time PCR analysis indicated that ac66 deletion did not affect the levels of viral DNA replication. Electron microscopy revealed that ac66 is not essential for nucleocapsid assembly, but for the efficient transport of nucleocapsids from the nucleus to the cytoplasm. These results suggested that ac66 plays an important role for the efficient exit of nucleocapsids from the nucleus to the cytoplasm for BV synthesis as well as for preoccluded virion and occlusion synthesis.

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1.

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2)copies/microl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.

Detection and analysis of horizontal gene transfer in herpesvirus.

Horizontal gene transfers, where a significant proportion of the coding DNA is contributed by external sources, might give rise to extremely dynamic genomes, which brings impact on the ecological and pathogenic characters of the recipient organisms. Therefore it is important to computationally discriminate between horizontal transferred genes and normal genes. In this paper, we introduce a novel method for identifying horizontal transferred genes. This method, which relies on a gene's nucleotide composition and hence obviates the need for knowledge of codon boundaries, is able to detect the horizontal transferred genes with an accuracy of higher than 90% within a reasonable length of time by using just a common PC. With this method, 141 putative transferred genes in mammalian herpesvirus were identified. Among them, 16 genes had been predicted or reported to have cellular homologues in previous papers, including those involved in immune systems and apoptosis such as GCR in EHV-2, BCL-2 (Bcelllymphoma/leukemia-2) homologue in MuHV-4, etc., and had been suggested being acquired from other organisms. Other 125 genes were identified for the first time. Twelve of the newly identified putative transferred genes had also been reported to participate in immune response, apoptosis, cell proliferation control or virulence determinant. Moreover, 42 of the 141 putative transferred genes were found to have non-virus homologues and so were convincingly revealed as transferred genes. Nine of the 42 transferred genes were phylogenetically analyzed, the origin and the relative origin time of which were discussed.

Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris using the GAP promoter.

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml(-1) and a cell wet weight of ca. 500 g l(-1) at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.

Identification of interspecies recombination among hepadnaviruses infecting cross-species hosts.

Members of the family Hepadnaviridae are divided into two genera, Orthohepadnavirus (from mammalian) and Avihepadnavirus (from avian). Recombination had been found to occur among human hepatitis B virus (HBV) strains of different genotypes, or between hepadnavirus strains from human and nonhuman primate. To reach a comparatively complete inspection of interspecies recombination events among hepadnavirus strains from various hosts, 837 hepadnavirus complete genome sequences from human and 112 from animals were analyzed by using fragment typing to scan for potential interspecies recombinants. Further bootscanning and phylogenetic analyses of the potential recombinants revealed six genome sequences as interspecies recombinants. Interspecies recombination events were found to occur among HBV strains from human and nonhuman primates, from gibbons of different genera, from chimpanzee and an unknown host, and between two avian hepadnavirus strains from birds of different subfamilies, which was identified for the first time. HBV interspecies recombinants were found to have recombination hot spots similar to that of human HBV intergenotype recombinants, breakpoints frequently locating near gene boundaries. Interspecies recombination found in this study may alter current views on hepadnavirus host specificity.

Characterization of a human telomerase reverse transcriptase sequence containing two antigenic epitopes with high affinity for human leucocyte antigen.

Almost all human malignant tumours exhibit strong telomerase activity, but normal adult tissues, with a few exceptions, do not. hTERT (human telomerase reverse transcriptase) is an essential component of telomerase, and hence it can serve as a parallel sign in the diagnosis and prognosis of cancers. In the present study, we selected a sequence of hTERT containing two antigenic epitopes that have high affinity for HLA-A2 (human leucocyte antigen-A2) as a TAA (tumour-associated antigen) based on a peptide-motif scoring system. The sequence was obtained by reverse-transcriptase PCR and cloned into the Escherichia coli expression vector pGEX-4T-1. The expression product appeared in the form of inclusion bodies. Denatured inclusion-body extract was subjected to SDS/PAGE, and the gel band corresponding to the putative 38 kDa fusion protein (GST-hTERT major tumour-associated antigen) was excised, ground with PBS, mixed with Freund's adjuvant and used to inoculate mice, generating anti-TERT polyclonal antibodies. Western blotting using the leukaemia cell line THP-1 demonstrated that the antibodies were able to detect hTERT expression, implying the potential applicability of the antigenic peptides derived from hTERT as a universal marker in the diagnosis and prognosis of tumours.